The long-term goal of this research project is to delineate the molecular mechanisms involved in controlling male reproduction. One of the major advances we have made recently is to provide the first evidence that GATA-1 factor, a GATA-binding protein which was originally considered as an erythroid cell-specific transcription factor, plays an important role in regulating the transcription of testicular genes including inhibin/activin alpha- and beta-B-subunit genes in MA-10, a mouse Leydig tumor cell line. We also demonstrated that another testicular GATA-binding protein, GAT-4, can selectively transactivate inhibin/activin beta-B-subunit gene. In view that the mechanisms by which GATA-binding protein(s) interacts and transactivates testicular genes have never been investigated, in this application we propose to use inhibin/activin alpha- and beta-B- subunit genes as mosel systems to study the actions of GATA-1 and GATA-4 on the control of the expression of specific genes in the testis. Specific Aim 1 is to investigate the mechanisms by which GATA-1 interacts and transactivates the testicular rat inhibin alpha- subunit gene in MA-10 and rat Sertoli cells, to examine the functional domains of GATA-1, and to identify protein factors that confer the testis-specific transactivaion of the alpha-subunit gene by GATA-1. Specific Aim 2 is to compare the mechanisms by which GATA-1 and GATA-4 upregulate the promoter for. the 4.8-kb rat inhibin/activin beta-B-subunit gene transcript, to identify the GATA and GATA-like sequences in the beta-B-subunit promoter that are responsible for the transactivation by GATA-1 and/or GATA-4 factor in testicular cells, and to determine the functional domains of GATA-4 that interact and transactivate the promoter for the 4.8-kb beta-B-subunit transcript and compare with those of GATA-1 factor. Specific Aim 3 is to investigate the developmental and hormonal regulation of GATA- binding proteins in rat testis and compare to those previously observed in inhibin/activin alpha- and beta-B-subunit genes. Specific Aim 4 is to isolate and characterize new testicular GATA-binding protein(s) that transactivates alpha- and/or beta-B-subunit promoters. The observations obtained from the proposed studies may yield some new information on the delineation of the mechanisms involved in the controls of testicular physiology.